A microarray starts with a piece of glass, or sometimes a silicon chip, the size of a microscope slide or smaller. Onto this substrate are fixed thousands of patches of single-stranded deoxyribonucleic acid (DNA), called probes, each patch measuring just tens of micrometers across. The location and sequence of each patch of DNA are known ahead of time. Genes are segments of DNA that contribute to phenotype/function. The function of genes is encoded in the sequence of chemicals that make up DNA. These chemicals, called nucleotides, each contain a sugar and phosphate backbone plus one of four molecules called bases-adenine (A), thymine (T), cytosine (C), or guanine (G). In the DNA molecule, the nucleotides are chemically bonded to and stacked atop each other in two strands forming a twisted ladder. The rungs of the ladder are the bases, with adenine always across from thymine and guanine always across from cytosine; however, under certain conditions the DNA helix can detach along the rungs of the ladder to form two single strands.
Microarrays draw on the so-called hybridization reaction. Two lengths of single-stranded DNA will bind together, or hybridize, only if the bases on one strand find a complementary sequence of bases on the other strand. In practice, every adenine base must match up with a thymine, and each guanine with a cytosine. A leading use of DNA microarrays is in determining which subset of a cell's genes are expressed, or are actively making proteins, under certain conditions, like exposure to a drug or toxic substance. A toxicology experiment, for example, might compare a normal cell to one that had been exposed to a drug. In a diagnostic experiment, such as in leukemia studies, two types of cancer cells are used.
The experiment works because when a cell is making protein, the DNA that specifies which type of protein to make is first transcribed into ribonucleic acid, or RNA—a chemical that can encode the same information as DNA. In the lab, scientists extract the RNA and then build single-stranded DNA copies of it. To aid in detecting the DNA on the microarray, they bind fluorescent molecules, or tags, into the new DNA. When the tagged single-stranded DNA is washed over the array, it sticks fast only to any single-stranded probe DNA that has a complementary gene-sequence to its own. Those with no complementary site on the array will wash off. A scan of the array with a laser or other excitation source causes any DNA that has found a tagged match to fluoresce, and that glow is picked up by a photodetector. The photodetector may consist, for example, of a charge-coupled device or a photomultiplier tube.
The fluorescent image of the microarray from the photodetector is then transmitted to a computer, which analyzes the location, color, and brightness of each patch of DNA on the microarray. Because the sequence of the array's DNA in each spot is known, the sequence of any DNA captured on that spot is also known. Comparing the colors found at those points on the array reveals the difference in gene expression between the two cells. Genetic variations called single nucleotide polymorphisms (SNPs) can also be uncovered by microarrays. SNPs are variations or mutations at a single spot in a gene's sequence. Since single-stranded DNA prefers to hybridize only with its perfect complement, arrays can determine the presence of such a mutation. SNPs are thought to be the key to understanding why people vary in their susceptibility to diseases.
Experimental arrays containing partial genomes of organisms such as yeast and humans are the bread-and-butter of most biotech firms, but many companies also custom-build arrays from gene sequences that customers upload to them. However, these processes are extremely expensive and time-consuming.
There are a number of ways that are conventionally known to make arrays. Affymetrix Inc., Santa Clara, Calif. makes high-density arrays with a method familiar to anyone in the semiconductor industry, namely, photolithography. This technology, known in the microarray industry as light-directed in situ synthesis, builds DNA probes one base/nucleotide at a time right on the chip (known also as a gene chip, or biochip). Construction begins with a glass slide that has been chemically primed with sites ready to bind nucleotides. The sites are capped by a photosensitive chemical that detaches under illumination. Light is shone through a patterned mask onto the chip, causing the capping chemical to break away from the areas it illuminates, thus exposing the primed spots. A solution containing one of the four types of nucleotides (each molecule of which is itself attached to a capping molecule) is then washed over the chip. The nucleotides bond only to the areas that have been exposed, and add a capping layer themselves. As the process can be repeated with another mask and a different nucleotide, a variety of DNA sequences can be built on the chip. Photolithography offers the highest density of probes per unit area of any technique currently in use. Production-scale chips can pack 400,000 probes in 20-μm patches. The single-stranded DNA reaches only 25 nucleotides in length, so that it takes several such patches to positively identify a single gene. One weakness to the current photolithography method is that a new set of masks must be produced for every new type of array. To overcome this problem, scientists at the University of Wisconsin in Madison, the University of Texas Southwestern Medical Center in Dallas, and Xeotron Corp. in Houston have demonstrated a maskless technique that uses an array of micromirrors that reflect onto the appropriate spots on the chip.
Perhaps the most straightforward array-making method is contact printing. A pin is first dipped into a solution containing pieces of DNA of uniform sequence that have been synthesized in the lab. The pin is pressed to the array surface, leaving behind a droplet of solution. Researchers and companies have developed several variations on this basic technique. The most obvious is the replicator pin, whose point must be rewetted after each deposition. Alternatively, pins with a split tip or a hollow tip hold a reservoir of fluid. In a third method, utilized by an acquired division of Affymetrix, a pin passes through a ring near its tip before contacting the array surface. The ring, once dipped into a solution of DNA, acts as a reservoir for the pin.
Corning Inc., of Corning, N.Y. chose the simple replicator pin design but executed it in a different manner. It prints a thousand spots of DNA simultaneously onto a glass microarray, far more than any other contact printing method. To break into high-density microarrays, Corning developed a printing system using a technique intended for printing color filters onto LCDs and applied it to building an array of DNA-printing pins. That technology involved a type of photosensitive glass into which features on the order of 100 μm can be etched. A pattern is projected onto the glass, which is then doused with hydrofluoric acid. The process yields a print head with 1000 pins about 100 μm in diameter, each separated by 100-120 μm. Figuring out how to wet each pin with a unique DNA sample was another challenge. The process utilizes a funnel-shaped reservoir of 1000 tightly packed conical cells. To make an array, a high-precision robotic system dips the pin head into the tip of the reservoir and then places it onto a glass slide. Corning currently uses a series of ten 1000-pin heads and reservoirs to produce arrays with 10,000 features each.
Inkjetting (Agilent Technologies Inc., headquartered in Santa Clara, Calif.) has two capabilities: it can print spots of DNA sequences synthesized in the lab and also, in a process called in situ fabrication, it can build up parts of genes on the array one base/nucleotide at a time. The inkjet technology is essentially the same as that found in a desktop printer. Jets of fluid are pressed through nozzles and broken into uniform droplets by the print head. For in situ synthesis, the four colors of ink—cyan, magenta, yellow, and black—are replaced with nucleotides of DNA having the four types of bases—adenine, guanine, cytosine, and thymine. The first set of nucleotides are ejected from the nozzle onto the substrate, then chemically fixed to the surface. The next set of nucleotides are jetted onto the first and chemically fixed to those. The process is repeated until the desired set of DNA is complete. This system can build lengths of DNA up to 60 nucleotides long. Of particular help is that different in situ arrays can be synthesized without a change in hardware or chemicals. In contrast, photolithographic methods require a set of masks for each new pattern.
The 60-base length is much shorter than an entire gene, which often runs to hundreds or thousands of bases. But using algorithms, Agilent claims that it can take a gene sequence and find a single 60-base sequence within the gene that will effectively identify it for genomics applications. In contrast to the in situ method, depositing pre-synthesized DNA uses about 100 jet nozzles, each spitting out a unique sequence of DNA. For both methods, the spot size ends up being 70-120 μm in diameter, allowing for arrays with about 25,000 features.
Electric fields have been found useful in microarrays. Motorola Inc. (Schaumburg, Ill.) is working on a method of detecting hybridized DNA using electrical signals rather than optical ones. The technology is incompatible with the glass arrays the company is developing because it requires addressable electrodes and other embedded circuitry. So Motorola has begun designing a silicon-based technology to take advantage of its semiconductor manufacturing experience. It plans to combine that experience with technology from the acquired Clinical Micro Sensors, Pasadena, Calif. which has developed a process that detects hybridization through a change in conductance.
Nanogen Inc. (San Diego, Calif.) is hoping the improved hybridization speed of its chip will give it a leg up in the medical diagnostics market. Companies envision microarrays that can detect the presence of genetic variations that make one drug therapy more efficacious than another. Some chips are already being tested in clinical laboratories, but estimates of when microarray-aided diagnostics will take off vary. For them to become an important market, the technology will have to improve. In particular, sample preparation time and complexity will have to decrease, so firms expect their current arrays to evolve into more complete on-chip laboratories capable of performing all the necessary procedures to extract genetic material from tissue or blood samples and then analyze it as well. The research market is shifting as well. As more and more DNA sequences are completed, biologists are looking downstream of DNA for clues to how the body works. And so their attention is turning to understanding proteins and their interactions. Some companies such as Ciphergen Biosystems Inc., of Fremont, Calif. are building their business around arrays of chemicals such as antibodies that will bind and identify proteins much as the DNA devices do. Several firms, including Agilent and Corning, believe their basic microarray platform will be compatible with producing such arrays. And one firm, Packard Instrument Co., of Meriden, Conn. has given up its DNA array business in favor of protein chips. Both protein chips and DNA arrays may benefit of the present invention described in detail below.
The above attempts at fabrication and utilizing microarrays have been found to suffer from various drawbacks. For example, the conventional high density microarrays (see Affymetrix Inc and Corning Inc. mentioned above) are limited by their current manufacturing processes. By shrinking the array's features, more functional chips may be made out of, for example, a single glass wafer. Making fewer wafers cuts down on the expensive chemicals needed to make the microarrays, and making smaller arrays reduces the amount of reagents needed to perform experiments with them. Still, scientists want more densely packed arrays. Ideally, they'd like to fit all of a human or other organism's genes onto a single microarray, so they can be studied all at once, rather than piecemeal. Recent estimates of the number of genes in a human genome may bring the goal posts a little closer. Scientists now say we have only 30 000-40 000 genes. Other drawbacks that exist with conventional microarrays, as mentioned above, include extremely high equipment and processing costs, lengthy microarray and gene probe fabrication times, inability to perform real-time gene probe programmability (customization), time-consuming and labor intensive gene detection protocols, high false-positive error rates due, for example, to poor probe quality, short resultant probe lengths, and bulkiness/non-portability of equipment.
It is therefore desirable to provide an array biochip system having ultra high-density capabilities that may optionally be employed in, for example, non-optical detection schemes, and that does not suffer from the above drawbacks.
These and other advantages of the present invention will become more fully apparent from the detailed description of the invention hereinbelow.